amino acid sequences for different strains of the organism came out 100% identical
just use SCHU S4
Etiologic Risk Group (see link below): Risk Group 3
Background/Disease Information:
-also known as the "rabbit fever"
-bacterium found in animals (especially rodents, rabbits & hares)
-spread by: getting infected insect bites, handling infected sick or dead animal, eating/drinking contaminated food/water, or inhaling airborne bacteria
-not known to spread from person to person
-can be treated with antibiotics
-symptoms: sudden fever, chills, headaches, diarrhea, muscle aches, progressive weakness
-about 400 cases in the United States every year
-10-50 organisms can cause the disease/ highly infectious http://www.bt.cdc.gov/agent/tularemia/facts.asp
Assay mixtures (200 µL): contained 100 mM Tris pH 7.8, 25 mM MgCl2, 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT)
Reagents: To determine cation specificity, assays were performed with 25 mM MgCl2, CaCl2, CoCl2, CuCl2, MnCl2, ZnCl2, or NiCl2
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
1-deoxy-D-xylulose 5-phosphate reductoisomerase from Yersinia pestis (3IIE)
Amino Acid Sequence: >fid|1288049|locus|FTT1574c| 1-deoxy-D-xylulose 5-phosphate reductoisomerase [Francisella tularensis subsp. tularensis SCHU S4] MFKKTKITILGATGSIGDSTLAVIRETNDFEVFALTAFSNVEKLAELCQEFKPKFAVVPD LSKKQKLQSLVTDVEVLVGESGLEKVSSLAEIDIVMSAIVGIAGLKPTFAAAKAGKKILL ANKESLVTAGHLLIDEVVKNNAQLIPVDSEHNAIFQCIDNHDKKCLPEIDKIILTASGGP FRDKQLHELTDVTPEQACNHPNWQMGRKISVDSSTMVNKALEVIEAYWLFSVSADKIGVL IHPQSVTHSMVRYVDGSYIAQLGVPDMKTPIANAMYYPKRGSVNVESLDFTKYQLTFREA CFERFEALKIVFNNLQNKNYAANIVFNAANEELVAAFLNKKIKYLEIIEVNKKVTKELNF ENPKNIEEVFEIDRKTREYVDSVLG length of your protein in Amino Acids 385
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 42847.4kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 24660 <Abs 0.1% (=1 g/l) 0.576, assuming all pairs of Cys residues form cystines>
Ext. coefficient 24410 <Abs 0.1% (=1 g/l) 0.570, assuming all Cys residues are reduced)>
NCBI Gene # or RefSeq#:
3191554
Protein ID (NP or XP #) or Wolbachia#: YP_170496.1
https://www.ncbi.nlm.nih.gov/protein/YP_170496.1
Organism: Francisella tularensis subsp. tularensis SCHU S4
Etiologic Risk Group (see link below): Risk Group 3
Background/Disease Information:
-also known as the "rabbit fever"
-bacterium found in animals (especially rodents, rabbits & hares)
-spread by: getting infected insect bites, handling infected sick or dead animal, eating/drinking contaminated food/water, or inhaling airborne bacteria
-not known to spread from person to person
-can be treated with antibiotics
-symptoms: sudden fever, chills, headaches, diarrhea, muscle aches, progressive weakness
-about 400 cases in the United States every year
-10-50 organisms can cause the disease/ highly infectious
http://www.bt.cdc.gov/agent/tularemia/facts.asp
Essentiality of this protein:
http://www.ncbi.nlm.nih.gov/pubmed?term=(1-deoxy-D-xylulose%205-phosphate%20reductoisomerase)%20AND%20Francisella%20Tularensis
Complex of enzymes:
EC#: 1.1.1.267
Link to BRENDA EC# page:
http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.1.267&Suchword=&organism%5B%5D=Francisella+tularensis&show_tm=0
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
- MEP synthase activity was evaluated at 22°C by spectrophotometrically monitoring the enzyme catalyzed oxidation of NADPH using an assay derived from that described by Takahashi et al.(http://www.ncbi.nlm.nih.gov/pubmed/9707569, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC21430/?tool=pubmed)
- Assay mixtures (200 µL): contained 100 mM Tris pH 7.8, 25 mM MgCl2, 0.15 mM NADPH, 7 µM MEP synthase, and a variable concentration of DXP (Echelon Biosciences, Salt Lake City, UT)
- Reagents: To determine cation specificity, assays were performed with 25 mM MgCl2, CaCl2, CoCl2, CuCl2, MnCl2, ZnCl2, or NiCl2
-- link to Sigma (or other company) page for assay or assay reagents (substrates)- 1-Deoxy-D-xylulose-5-phosphate sodium salt(13368 SIGMA): 1MG - $66.30 / 5MG-$264.50
(http://www.sigmaaldrich.com/catalog/product/sigma/13368?lang=en®ion=US)(http://www.sigmaaldrich.com/catalog/product/sigma/n6505?lang=en®ion=US)
-- link to paper that contains assay information
from article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788227/?tool=pubmed
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if homology model:
3IIE_A
http://www.pdb.org/pdb/explore/explore.do?structureId=3IIE
-- Query Coverage (if not direct match): 98%
-- Max Ident (if not direct match): 50%
Druggable Target (see Databases for this):
Current Inhibitors: fosmidomycin
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788227/?tool=pubmed
Expression Information (has it been expressed in bacterial cells):
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788227/?tool=pubmed
http://pubs.acs.org/doi/full/10.1021/bi047312p for E.coli
Purification Method:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788227/?tool=pubmed
http://pubs.acs.org/doi/full/10.1021/bi047312p for E.coli
Image of protein (PyMol or etc):
Amino Acid Sequence:
>fid|1288049|locus|FTT1574c| 1-deoxy-D-xylulose 5-phosphate reductoisomerase [Francisella tularensis subsp. tularensis SCHU S4]
MFKKTKITILGATGSIGDSTLAVIRETNDFEVFALTAFSNVEKLAELCQEFKPKFAVVPD LSKKQKLQSLVTDVEVLVGESGLEKVSSLAEIDIVMSAIVGIAGLKPTFAAAKAGKKILL ANKESLVTAGHLLIDEVVKNNAQLIPVDSEHNAIFQCIDNHDKKCLPEIDKIILTASGGP FRDKQLHELTDVTPEQACNHPNWQMGRKISVDSSTMVNKALEVIEAYWLFSVSADKIGVL IHPQSVTHSMVRYVDGSYIAQLGVPDMKTPIANAMYYPKRGSVNVESLDFTKYQLTFREA CFERFEALKIVFNNLQNKNYAANIVFNAANEELVAAFLNKKIKYLEIIEVNKKVTKELNF ENPKNIEEVFEIDRKTREYVDSVLG
length of your protein in Amino Acids 385
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 42847.4kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 24660 <Abs 0.1% (=1 g/l) 0.576, assuming all pairs of Cys residues form cystines>
Ext. coefficient 24410 <Abs 0.1% (=1 g/l) 0.570, assuming all Cys residues are reduced)>
CDS Gene Sequence:
GC% Content for gene: 8.94%
CDS Gene Sequence (codon optimized) - after having done Primer Design:
GC% Content for gene (codon optimized): 48.22%
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
ATGTTCAAAAAAACCAAAATCACCATCC
CCAGGGTAGAGTCACCGATAGAACCGGTCGCACCCAGGATGGTGATTTTGGTTTTTTTGA
TATCGGTGACTCTACCCTGGCGGTTATCCGTGAAACCAATGATTTCGAAGTTTTCGCGCT
GCACAGTTCCGCCAGTTTCTCAACGTTAGAGAACGCGGTCAGCGCGAAAACTTCGAAATC
AACTGGCGGAACTGTGCCAGGAATTCAAACCGAAATTTGCGGTTGTTCCTGATCTGTCTA
CTTCAACGTCGGTAACGAGAGACTGGAGTTTCTGTTTTTTAGACAGATCAGGAACAACCG
CTCTCGTTACCGACGTTGAAGTGCTGGTTGGTGAATCTGGTCTGGAAAAAGTTTCTTCTC
CCAACGATCGCAGACATAACAATGTCGATCTCAGCCAGAGAAGAAACTTTTTCCAGACCA
GTTATGTCTGCGATCGTTGGTATCGCGGGTCTGAAACCGACCTTTGCGGCAGCGAAAGCG
GGCGGTAACCAGAGACTCTTTGTTCGCCAGCAGGATTTTCTTACCCGCTTTCGCTGCCGC
AGAGTCTCTGGTTACCGCCGGTCATCTGCTGATCGACGAAGTTGTTAAGAACAACGCCCA
TGGAAGATCGCGTTGTGTTCAGAATCCACCGGGATCAGTTGGGCGTTGTTCTTAACAACT
AACACAACGCGATCTTCCAGTGCATTGACAACCACGACAAAAAATGCCTGCCGGAAATCG
TTGTCACGGAACGGACCACCAGACGCAGTCAGGATGATTTTATCGATTTCCGGCAGGCAT
TGGTCCGTTCCGTGACAAACAGCTGCACGAACTGACGGACGTTACCCCAGAGCAGGCGTG
AGTCGACAGAGATTTTACGACCCATCTGCCAGTTCGGGTGATTACACGCCTGCTCTGGGG
GGTCGTAAAATCTCTGTCGACTCTTCTACGATGGTAAACAAAGCCCTGGAAGTTATCGAA
ACCGATTTTGTCCGCAGAAACAGAGAACAGCCAGTAAGCTTCGATAACTTCCAGGGCTTT
TTCTGCGGACAAAATCGGTGTTCTGATCCACCCGCAGTCTGTTACCCACTCTATGGTTCG
GGGACACCGAGTTGAGCAATATAAGAGCCGTCTACGTAACGAACCATAGAGTGGGTAACA
TGCTCAACTCGGTGTCCCTGACATGAAAACCCCGATTGCGAACGCCATGTACTATCCTAA
TCGTGAAGTCCAGGGATTCAACATTAACAGAACCACGTTTAGGATAGTACATGGCGTTCG
TGAATCCCTGGACTTCACGAAGTACCAGCTGACCTTCCGCGAAGCGTGCTTCGAACGTTT
TTTTTGTTCTGCAGGTTGTTGAAAACAATTTTCAGAGCTTCAAAACGTTCGAAGCACGCT
TCAACAACCTGCAGAACAAAAACTACGCGGCCAACATCGTATTCAACGCCGCGAACGAAG
TCCAGGTATTTGATCTTCTTGTTCAGGAAGGCCGCAACCAGTTCTTCGTTCGCGGCGTTG
GAACAAGAAGATCAAATACCTGGAGATCATTGAAGTAAATAAGAAGGTTACCAAAGAGCT
AGACTTCCTCAATGTTTTTAGGGTTCTCGAAATTCAGCTCTTTGGTAACCTTCTTATTTA
CCCTAAAAACATTGAGGAAGTCTTCGAGATTGACCGTAAAACGCGTGAGTATGTGGACTC
TTAGCCCAGAACAGAGTCCACATACTCACGCG
Primer design results for 'tail' primers (this is just 2 sequences):
Link to Final Results Logfile
http://helixweb.nih.gov/tmp/dnaworks_1338926014p1697_2055/LOGFILE.txt
Upstream
TACTTCCAATCCATGTTCAAAAAAACCAAAATC
Downstream
TATCCACCTTTACTGTTAGCCCAGAACAGAGTC
>filtered DNA sequence consisting of 1158 bases
>filtered DNA sequence consisting of 1158 bases. ATGTTCAAAAAAACCAAAATCACCATCCTGGGTGCGACCGGTTCTATCGGTGACTCTACC CTGGCGGTTATCCGTGAAACCAATGATTTCGAAGTTTTCGCGCTGACCGCGTTCTCTAAC GTTGAGAAACTGGCGGAACTGTGCCAGGAATTCAAACCGAAATTTGCGGTTGTTCCTGAT CTGTCTAAAAAACAGAAACTCCAGTCTCTCGTTACCGACGTTGAAGTGCTGGTTGGTGAA TCTGGTCTGGAAAAAGTTTCTTCTCTGGCTGAGATCGACATTGTTATGTCTGCGATCGTT GGTATCGCGGGTCTGAAACCGACCTTTGCGGCAGCGAAAGCGGGTAAGAAAATCCTGCTG GCGAACAAAGAGTCTCTGGTTACCGCCGGTCATCTGCTGATCGACGAAGTTGTTAAGAAC AACGCCCAACTGATCCCGGTGGATTCTGAACACAACGCGATCTTCCAGTGCATTGACAAC CACGACAAAAAATGCCTGCCGGAAATCGATAAAATCATCCTGACTGCGTCTGGTGGTCCG TTCCGTGACAAACAGCTGCACGAACTGACGGACGTTACCCCAGAGCAGGCGTGTAATCAC CCGAACTGGCAGATGGGTCGTAAAATCTCTGTCGACTCTTCTACGATGGTAAACAAAGCC CTGGAAGTTATCGAAGCTTACTGGCTGTTCTCTGTTTCTGCGGACAAAATCGGTGTTCTG ATCCACCCGCAGTCTGTTACCCACTCTATGGTTCGTTACGTAGACGGCTCTTATATTGCT CAACTCGGTGTCCCTGACATGAAAACCCCGATTGCGAACGCCATGTACTATCCTAAACGT GGTTCTGTTAATGTTGAATCCCTGGACTTCACGAAGTACCAGCTGACCTTCCGCGAAGCG TGCTTCGAACGTTTTGAAGCTCTGAAAATTGTTTTCAACAACCTGCAGAACAAAAACTAC GCGGCCAACATCGTATTCAACGCCGCGAACGAAGAACTGGTTGCGGCCTTCCTGAACAAG AAGATCAAATACCTGGAGATCATTGAAGTAAATAAGAAGGTTACCAAAGAGCTGAATTTC GAGAACCCTAAAAACATTGAGGAAGTCTTCGAGATTGACCGTAAAACGCGTGAGTATGTG
GACTCTGTTCTGGGCTAA
Upstream
33bp
GC Content: 30.3%
Downstream
33bp
GC Content: 45.5%